Single-cell transcriptomics reveal cellular diversity of aortic valve and the immunomodulation by PPARγ during hyperlipidemia

Figures

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  Fig. 1. LDL is a primary lipoprotein accumulated in the aortic valve under hyperlipidemia. a–d Comparison of lipid accumulation in the aortic valves and blood lipid profiles of mice (C57BL/6J, Apoe−/−, and Ldlr−/−) fed a chow diet (chow) versus a western diet (WD) for 10 weeks (n = 5). Representative lipid stain images and measurement of valvular lipid deposition. Black or dark brown spots are melanin pigments. Arrowhead: accumulated lipids. Scale bar: 150 μm (left), 30 μm (right) (a), total cholesterol and LDL levels in the blood plasma (b), correlations between aortic valvular lipid deposition and blood lipid profiles of WD-fed hyperlipidemic mice (c), and correlation between aortic valvular lipid accumulation and aortic sinus lesions in WD-fed hyperlipidemic mice (d). e, f DiI-lipoproteins (LDL or VLDL) uptake of aortic valves cultured ex vivo. Representative whole-mount images (repeated three times) (e) and flow cytometry analysis (n = 4) (f). Scale bar: 100 μm. g DiI-lipoprotein (LDL or VLDL) uptake levels of cultured VICs using C57BL/6J, Ldlr−/−, and Apoe−/− mice. Representative images (top) and mean fluorescence intensity (MFI) of DiI-lipoprotein detected by flow cytometry (n = 2.…

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  Fig. 2. Plasma LDL levels positively correlate with inflammation and lipid accumulation in the aortic valve. a–c Flow cytometry analyses of the aortic valves of C57BL/6 J, Apoe−/−, and Ldlr−/− mice fed a chow diet (chow) or western diet (WD) for 10 weeks (n = 5). Representative plot of leukocytes (a), percentage of leukocytes, macrophages, and macrophage subsets (b), and correlations between the percentage of MHC-IIhi CD11c+ CD206- macrophages and blood lipid profiles of WD-fed hyperlipidemic mice (c). d–i Apoe−/− mice were injected with PCSK9-AAV to identify the effect of elevated serum LDL levels. PCSK9-AAV-injected Apoe−/− mice were compared with non-injected Apoe−/− mice (WD for 24 weeks). Representative Oil Red O stain images. Scale bar: 150 μm (top), 50 μm (bottom) (d), quantification of valvular lipid deposition and thickness of aortic valve (n = 5) (e), total cholesterol and LDL levels in blood plasma (n = 5 for Apoe−/−, n = 4 for Apoe−/−+PCSK9-AAV) (f), correlation between valvular lipid deposition and each lipid profile (total cholesterol and LDL) (n = 9 total; 5 from Apoe−/−; 4 from Apoe−/−+PCSK9-AAV) (g), immunostaining of CD68 (red) and Vimentin (white) along with the lipid…

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  Fig. 3. Comprehensive single-cell profiling reveals that monocyte-derived macrophages are the major immune cells accumulated in aortic valve during hyperlipidemia. a Uniform manifold approximation and projection (UMAP) plot of 6574 single cells colored by the clusters (left) and mouse models (right). Apoe−/− and Ldlr−/− mice were fed a WD for 16 weeks. b Average expression map of known cell-type marker genes for each cell cluster. Color represents average expression levels, which are scaled by z-transformation and limited to a scale from −2.5 to 2.5. Dot size represents the fraction of cells with the expression value of each marker gene for each cluster. c Absolute cell number (left) and relative proportion (right) of the major cell lineages from each mouse model. d UMAP plot of gene expression of H2-Ab1, Itgax, and Mrc1 (gray to blue). e Whole-mount immunostaining of the aortic valve from C57BL/6J, Apoe−/− and Ldlr−/− mice for MHC-II (red) and CD206 (green). Scale bar: 100 μm. f Accumulated MHC-II+ cells in the aortic valve of PCSK9-AAV-injected Ccr2+/+ or -Ccr2−/− mice (WD for 24 weeks). Representative whole-mount MHC-II immunofluorescence images (left) and measurement of MHC-II +…

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  Fig. 4. Pro-inflammatory valvular macrophages are markedly increased during hyperlipidemia. a UMAP plot of 3160 leukocytes, colored by the clusters and mouse models as indicated. Apoe−/− and Ldlr−/− mice were fed a WD for 16 weeks. b Average expression map of known cell-type marker genes for each cell cluster. Color represents average expression levels which are scaled by z-transformation and limited to a scale from −2.5 to 2.5. Dot size represents the fraction of cells with the expression value of each marker gene for each cluster. c Absolute cell number (left) and relative proportion (right) of leukocyte subsets from each mouse model. d UMAP plot of expression level of marker genes (gray to blue). e Trajectory component plot of 2677 macrophages colored by the cell states, clusters, and mouse models. f Expression map of top10 significant genes for each cell state. Color represents the expression levels, which are scaled by z-transformation and limited to a minimum scale of −2.5. (purple to yellow). g Correlation of Monocle pseudotime with functional features of macrophages. Pro-inflammatory score represents mean expression of featured genes: Il1b, Tnf, Ccl2, Cxcl10, Cxcl2, H2-Ab1, and…

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  Fig. 5. VICs show pro-inflammatory features during hyperlipidemia. a UMAP plot of 1,732 VICs colored by clusters and mouse models as indicated. b Absolute cell numbers (left) and relative proportions (right) of the VIC subsets from each mouse model. c Average expression map of representative genes in each cell cluster. Color depends on the mouse model, and size represents the fraction of cells with the expression value of each gene for each group. d, e Representative gene expression of VIC subclusters. UMAP plot of gene expression (gray to blue) (d) and RNA in situ hybridization (e). Scale bar: 50 μm. f Boxplot of average expression level of genes related to myofibroblast activation (left) and calcification (right) (Gene list in Supplementary Data 2) (n = 981 cells for C57BL/6J; 470 for Apoe−/−; 281 for Ldlr−/−). Each box depicts the IQR and median of each score, whiskers indicate 1.5 times the IQR. p, two-sided T-test p-value. g, h Expression map of monocyte chemoattractant genes. The average expression of genes for VICs (purple to yellow) in each mouse model was scaled by z-transformation and displayed on a scale of at least−2.5 (g) and RNA in situ hybridization of Csf1 and Cx3cl1 (h).…

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  Fig. 6. VECs contain three main subtypes and Cd36+ VECs are markedly increased in hyperlipidemic mice. a UMAP plot of 536 VECs colored by the clusters and mouse models as indicated. b UMAP plot of VECs color-coded by expression (gray to blue) of marker genes. c Pie chart for the relative proportion of the VEC subsets from each mouse model. d Expression of pro-inflammatory genes in VECs (VEC_C0, C1, and C2). Heatmap (left) and boxplot (right) of the average expression level of genes listed in the heatmap. Heatmap are displayed as expression values scaled by z-transformation on a scale of at least-2.5. Cells having no expression for pro-inflammatory genes were excluded (n = 274 cells for C57BL/6J; 88 for Apoe−/−; 80 for Ldlr−/−). Each box depicts the IQR and median of each score, whiskers indicate 1.5 times the IQR. p, two-sided T-test p-value. e Enrichment plot for significant Gene Ontology (GO) terms related to monocyte chemotaxis. Genes were ranked by the fold changes between knockout and wild-type models for cells in VEC clusters (VEC_C0, C1, and C2). f Identification of the localization of VEC subclusters using RNA in situ hybridization (Fgfr3 and Cd36) or immunofluorescence (PROX1).…

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  Fig. 7. PPARγ pathway is activated in VECs of hyperlipidemic mice and conserved in human aortic valves. a Average expression map of genes in PPARγ regulon, produced by Single-Cell Regulatory Network Inference and Clustering (SCENIC), for each cluster of leukocytes, VECs, and VICs. b UMAP plot of VECs color-coded by the activity of PPARγ regulon. Gene set activity was calculated by SCENIC. AUC: area under curve. c Immunostaining of PPARγ (red) and endomucin (EMCN, EC marker, green) in aortic valve with sinus from normal (chow diet) and hyperlipidemic mice (Apoe−/− and Ldlr−/− mice, WD for 16 weeks) (n = 4). DAPI (blue) was used to stain nuclei. The graph represents the relative MFI of PPARγ in the VECs. Kruskal–Wallis test with post-hoc Dunn’s test was used. Scale bar: 30 μm. d UMAP plot of 41,326 single-cells derived from human aortic valve, colored by the clusters (left) and samples (right). e Average expression map of genes in PPARγ regulon for each cell cluster from human aortic valve. f Pro-inflammatory (top) and cell adhesion molecule (bottom) scores in non-targeting siRNA (NC) and PPARG targeting siRNA-treated (PPARG knockdown, KD) human VECs under no (NT) and oxLDL treatment…

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  Fig. 8. PPARγ activation protects the aortic valve against the inflammation. a Adhesion assay of monocytes to ex vivo cultured aortic valve treated with oxLDL and/or inhibition of PPARγ by T0070907 (n = 7 for non-treated, n = 7 for oxLDL, n = 6 for oxLDL+T0070907). Scale bar: 50 μm. b, c Pro-inflammatory effect of T0070907 (PPARγ antagonist) on mouse aortic valve in vivo. Ldlr−/− mice were intraperitoneally injected daily with vehicle or T0070907 for 10 weeks with a WD feeding. Representative IHC images (b) and flow cytometry analysis presenting percentage of each immune cell subset (n = 6) (c). Scale bar: 30 μm. d Flow cytometry analysis showing anti-inflammatory effect of PPARγ activation by pioglitazone on mouse aortic valve in vivo. PCSK9-AAV-injected C57BL/6J mice were fed with pioglitazone-containing WD or normal WD for 6 weeks. Graphs present percentage of each immune cell subset (n = 10). e Proposed pathogenesis model of the early-stage aortic valve disease induced by hyperlipidemia. In hyperlipidemic states, oxidized LDL triggers aortic valvular inflammation by enhancing the productions of various cytokines and chemokines, leading to the recruitment of monocyte-derived MHC-IIhi…